Distinct p53 acetylation cassettes differentially influence gene-expression patterns and cell fate

The activity of the p53 gene product is regulated by a plethora of posttranslational modifications. An open question is whether such posttranslational changes act redundantly or dependently upon one another. We show that a functional interference between specific acetylated and phosphorylated residues of p53 influences cell fate. Acetylation of lysine 320 (K320) prevents phosphorylation of crucial serines in the NH2-terminal region of p53; only allows activation of genes containing high-affinity p53 binding sites, such as p21/WAF; and promotes cell survival after DNA damage. In contrast, acetylation of K373 leads to hyperphosphorylation of p53 NH2-terminal residues and enhances the interaction with promoters for which p53 possesses low DNA binding affinity, such as those contained in proapoptotic genes, leading to cell death. Further, acetylation of each of these two lysine clusters differentially regulates the interaction of p53 with coactivators and corepressors and produces distinct gene-expression profiles. By analogy with the “histone code” hypothesis, we propose that the multiple biological activities of p53 are orchestrated and deciphered by different “p53 cassettes,” each containing combination patterns of posttranslational modifications and protein–protein interactions

Macrophage LRP1 suppresses neo-intima formation during vascular remodeling by modulating the TGF-β signaling pathway.

Vascular remodeling in response to alterations in blood flow has been shown to modulate the formation of neo-intima. This process results from a proliferative response of vascular smooth muscle cells and is influenced by macrophages, which potentiate the development of the intima. The LDL receptor-related protein 1 (LRP1) is a large endocytic and signaling receptor that recognizes a number of ligands including apoE-containing lipoproteins, proteases and protease-inhibitor complexes. Macrophage LRP1 is known to influence the development of atherosclerosis, but its role in vascular remodeling has not been investigated.To define the contribution of macrophage LRP1 to vascular remodeling, we generated macrophage specific LRP1-deficient mice (macLRP1-/-) on an LDL receptor (LDLr) knock-out background. Using a carotid ligation model, we detected a 2-fold increase in neointimal thickening and a 2-fold increase in the intima/media ratio in macLRP1-/- mice. Quantitative RT-PCR arrays of the remodeled vessel wall identified increases in mRNA levels of the TGF-β2 gene as well as the Pdgfa gene in macLRP1-/- mice which could account for the alterations in vascular remodeling. Immunohistochemistry analysis revealed increased activation of the TGF-β signaling pathway in macLRP1-/- mice. Further, we observed that LRP1 binds TGF-β2 and macrophages lacking LRP1 accumulate twice as much TGF-β2 in conditioned media. Finally, TNF-α modulation of the TGF-β2 gene in macrophages is attenuated when LRP1 is expressed. Together, the data reveal that LRP1 modulates both the expression and protein levels of TGF-β2 in macrophages.Our data demonstrate that macrophage LRP1 protects the vasculature by limiting remodeling events associated with flow. This appears to occur by the ability of macrophage LRP1 to reduce TGF-β2 protein levels and to attenuate expression of the TGF-β2 gene resulting in suppression of the TGF-β signaling pathway

Correction: LDL Receptor-Related Protein-1 (LRP1) Regulates Cholesterol Accumulation in Macrophages.

Within the circulation, cholesterol is transported by lipoprotein particles and is taken up by cells when these particles associate with cellular receptors. In macrophages, excessive lipoprotein particle uptake leads to foam cell formation, which is an early event in the development of atherosclerosis. Currently, mechanisms responsible for foam cell formation are incompletely understood. To date, several macrophage receptors have been identified that contribute to the uptake of modified forms of lipoproteins leading to foam cell formation, but the in vivo contribution of the LDL receptor-related protein 1 (LRP1) to this process is not known [corrected]. To investigate the role of LRP1 in cholesterol accumulation in macrophages, we generated mice with a selective deletion of LRP1 in macrophages on an LDL receptor (LDLR)-deficient background (macLRP1-/-). After feeding mice a high fat diet for 11 weeks, peritoneal macrophages isolated from Lrp+/+ mice contained significantly higher levels of total cholesterol than those from macLRP1-/- mice. Further analysis revealed that this was due to increased levels of cholesterol esters. Interestingly, macLRP1-/- mice displayed elevated plasma cholesterol and triglyceride levels resulting from accumulation of large, triglyceride-rich lipoprotein particles in the circulation. This increase did not result from an increase in hepatic VLDL biosynthesis, but rather results from a defect in catabolism of triglyceride-rich lipoprotein particles in macLRP1-/- mice. These studies reveal an important in vivo contribution of macrophage LRP1 to cholesterol homeostasis

LRP1 expressed in macrophages protects against vascular remodeling in the carotid-ligation model.

<p>a) Photomicrographs showing the representative Elastic-Van Gieson staining of contralateral (<i>left panels</i>) and ligated vessels (<i>right panels</i>) in LRP1+/+ (<i>top</i>) and macLRP1-/- (<i>bottom</i>) mice. b) H&E staining of the serial sections of the same artery as in (a). c) Morphometric measurements of the total vessel area, adventitia area, media area and intima area were measured for LRP1+/+ (n = 20) and macLRP1-/- (n = 20) mice (*p = 0.03; **p = 0.009, Students <i>t</i>-test). d) Ratio of the Intima over the Media is represented for LRP1+/+ and macLRP1-/- mice (***p = 0.010, Students <i>t</i>-test).</p

Increased cell proliferation and PDGFR-βexpression during vascular remodeling.

<p>Representative anti-PDGFR-β staining (a,b,c) and anti-PCNA staining (d,e,f) for proliferating cells of contralateral (a,d) and ligated carotid arteries (b,c,e,f). Internal elastic lamina (IEL) is marked by a black arrow. Arrowheads indicate the cells positive for antibody staining. L identifies the lumen.</p

Effective deletion of the <i>Lrp1</i> gene in macrophages from macLRP1-/- mice.

<p>a) Bone marrow derived macrophages from LRP1+/+ and macLRP1-/- mice were analyzed for LRP1 expression by immunoblot analysis. b) Resident macrophages were isolated by peritoneal lavage and incubated with fluorescent labeled RAP for 1 h at 37 ^oC. RAP-positive, F4/80 positive cells were then determined by FACS analysis.</p

Evaluation of matrix deposition in neointima.

<p>Photomicrographs showing the representative Fibrin-Fraser-Lendrum stained sections of contralateral (a) and ligated vessels (b,c). Internal elastic lamina (IEL) is marked by black arrow. White asterisk show collagen matrix specific green color deposition. L identifies the lumen.</p

TGF-β2 induces SMAD and MAPK signaling in hAoSMCs and TGF-β-mediated ERK activation is inhibited in LRP1+/+ expressing macrophages.

<p>a) hAoSMCs were serum starved for overnight and induced with 2 ng/ml TGF-β2 at indicated times. Cell extracts were analyzed for phospho-SMAD2 (S465/467), phospho-Erk1/2 (T202/Y204) or α/β tubulin for loading control. Results are representative of two independent experiments. b,c) Bone marrow derived macrophages from LRP1+/+ or macLRP1-/- mice were treated with TGF-β2 (b) or TGF-β1 (c). Cell extracts were then analyzed for phospho-Erk1/2 and/or phospho-SMAD2 by immunoblot analysis. α/β tubulin levels represents loading control measured by immunoblot analysis.</p

Increased activation of the TGF-β signaling pathway during vessel remodeling in macLRP1-/- mice.

<p>Representative images from contralateral (a,d) and ligated vessels (b,c,e,f) stained for TGF-β2 (a,b,c) or phospho-Smad 2/3 (d,e,f). Internal elastic lamina (IEL) is marked by a black arrow. Arrowheads indicate a cell positive for antibody staining while L marks the lumen. (g) five fields from each mouse were assessed for the number of phospho-Smad2/3 positive nuclei for LRP1+/+ (n = 2 mice) and macLRP1-/- (n = 2 mice). *p<0.0001, students <i>t-</i>test.</p